Mammals have two known B12-dependent enzymes that conduct essential housekeeping functions: methionine synthase and methylmalonyl-CoA mutase. Impairments in methionine synthase lead to megaloblastic anemia and hyperhomocysteinemia, the latter being a graded risk factor for atherosclerotic cardiovascular diseases. Dysfunction of methylmalonyl-CoA mutase leads to methylmalonic aciduria, leading to metabolic aberrations that can be fatal. This proposal focuses on elucidating the reaction mechanisms of these two enzymes, as well as the mechanism by which the cofactor regulates the activity of methionine synthase. Chemically, B12 is a novel cofactor, and catalyzes distinct biochemical transformations in association with these two enzymes. Thus, methionine synthase catalyzes a displacement reaction in which the cobalt-carbon bond of B12 breaks heterolytically, while the mutase catalyzes a rearrangement reaction in which the cobalt-carbon bond breaks homolytically. A combination of spectroscopic, kinetic and molecular genetic techniques will be used to explore the mechanisms of these reactions, and to map and characterize the mutations in patients with inborn errors affecting these enzymes. The redox-active proteins that activate mammalian methionine synthase under physiological conditions will be identified and characterized, as these represent additional targets for mutations that could lead to hyperhomocysteinemia. The level at which B12 exerts control over methionine synthase and causes the observed induction in enzyme activity when presented to cells in culture will also be examined. These studies on the native and mutant B12 enzymes will make inroads into our understanding of novel reaction mechanisms of clinically important enzymes.